Association of CpG-SNP and 3'UTR-SNP of WFS1 with the Risk of Type 2 Diabetes Mellitus in an Iranian Population

Type 2 diabetes mellitus (T2DM) is one of the most common multifactorial disorders in Iran. Recent genome wide association studies (GWASs) and functional studies have suggested that WFS1 may predispose individuals to T2DM. However, to date, the possible association of such variants with T2DM in Iranians remained unknown. Here, we investigated the association of the two polymorphisms of WFS1 (rs1801214 a CpG-SNP, and rs1046320 a 3’UTR-SNP) with T2DM in an Iranian population. The study population comprised 432 unrelated Iranian individuals including 220 patients with T2DM, and 211 unrelated healthy control subjects. Genotyping was performed using PCR-RFLP, and confirmed with sequencing. In a logistic regression analysis, the rs1801214-T allele was associated with a significantly lower risk of T2DM assuming the log-additive model (OR: 0.68, 95% CI: 0.52-0.91, P= 0.007539). Moreover, the G allele of rs1046320 was associated with a lower risk of T2DM assuming the log-additive model (OR: 0.68, 95% CI: 0.50- 0.91, P= 0.008313). Haplotype analysis revealed that haplotypes that carry at least one protective allele are associated with a lower risk of T2DM. This is a first evidence for the association of WFS1 rs1801214, and rs1046320 with T2DM in an Iranian population.

14% in Iranians aged above 30 years, and according to the International Diabetes Federation (IDF) website, more than 4.5 million Iranians were diagnosed as diabetic cases in 2014 (1,2). Sufficient insulin secretion from pancreas beta cells is essential to maintain blood glucose within normal range, and destroy beta cells during T2D development upon insufficient insulin secretion and diabetes progression (3,4).
A variety of environmental, epigenetic, genetic factors. and also interplay between these factors contribute to the development of T2DM.
Genetic susceptibility to T2DM has been extensively explored by genome-wide association studies (GWASs), and accordingly a number of loci have been identified to be associated with T2DM (5)(6)(7). Among these, variants in the Wolferamin (WSF1) have been shown to modify the susceptibility to T2DM in different studies (8,9).
Wolferamin is a 890 amino-acid transmembrane polypeptide located on chromosome 4p16 that is ubiquitously expressed, but has high expression rate in specific neurons and pancreatic beta cells (10). This protein is a novel endoplasmic reticulum (ER) transmembrane glycoprotein that plays a role in regulating calcium fluxes, and hemostasis in the ER (11,12). Mutations in WFS1 are known to cause the Wolfram syndrome (WFS; OMIM 222300), an autosomal recessive neurodegenerative disorder that is clinically defined by diabetes insipidus, young-onset non-immune insulin-dependent diabetes mellitus, optic atrophy, and deafness. Therefore, WFS is also known as DIDMOAD syndrome (13). A growing body of evidence suggested that highly developed ER structure in beta cells had important roles in insulin production, and secretion in response to blood glucose levels (14)(15)(16)(17). Impaired insulin secretion due to epigenetic modifications, and pathogenic variants in the WSF1 results in progressive rodent and human glucose intolerance, and insulin deficiency due to ER stress and apoptosis of beta cells (11,18,19). Polymorphisms of the WFS1 was suggested as a minor modulator of gene function, and susceptibility to polygenic forms of diabetes.
To the best of our knowledge, there are no data regarding the possible contribution of the GWASidentified locus at this gene to T2DM in Iranian population.
In this study, we evaluated the association of two WFS1 variants with T2DM in an Iranian population. These variants include WFS1 rs1801214, an index variant of the GWAS identified locus that removes a cytosine-phosphateguanine (CpG) dinucleotide, and rs1046320 at 3'UTR of WFS1 that is a candidate functional variant in high linkage disequilibrium (r 2 = 0.97) with the index variant.

Subjects
The study population comprised 432 unrelated Iranian individuals, including 220 patients with T2DM and 211 unrelated healthy control subjects, matched for age and gender. The diagnosis of T2DM was based on the WHO criteria (20,21).  Table 2).

Statistical analysis
Genotyping results were tested for significant departure from Hardy-Weinberg equilibrium among patients and controls using χ 2 and all statistical analyses were performed by R programming language (version 3.1.0) (23 (Table 3).

Haplotype analysis
We further estimated the frequencies of haplotypes for these SNPs, and evaluated the association of haplotypes with T2DM. Table 4 represents the frequency of haplotypes in the studied population along with the results of association analysis. The most frequent haplotype (haplotype 1 in the table 4, consisting of rs1801214-C, and rs1046320-A) was used as a reference haplotype. The results showed that haplotypes that contain at least one protective allele (either rs1801214-T or rs1046320-G) had a lower risk of T2DM in comparison to the reference haplotype (Table 4).

Discussion
T2DM is one of the most worldwide health problems of the 21 th century, and many genetic variants were shown to be associated with disease susceptibility. WFS1 is a new susceptible diabetes gene, which has been confirmed recently with genome wide association, and replication studies (8,9,(24)(25)(26). We showed that rs1801214 was significantly associated with T2DM in the studied Iranian population. Moreover, this is the first replication study for this SNP and the results were in line with the original GWAS with the T allele being associated with a lower risk of T2DM (8).
Subjects with at least one T allele had a lower risk of T2DM. WFS1 rs1801214 is located in the coding  This protective effect was also confirmed in a metaanalysis performed by the same authors (24).
Another study on Hungarian population has evaluated the association of WFS1 3'UTR variants potentially affecting miRNA binding including rs1046320 with T2DM, and confirmed the protective role of the G allele (25). Besides, rs1046320 may potentially alter miR-204-5p and miR-211-5p binding to WFS1 transcript that may imply a mechanism by which this SNP contribute to T2DM risk.